Plant sRNA extract or plant miRNA for use as an immunosuppressive agent

ABSTRACT

The present invention relates to a plant sRNA extract for use as an immunosuppressive agent and to plant miRNA for use as an immunosuppressive agent.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is continuation of U.S. application Ser. No.15/307,119, filed Oct. 27, 2016, now U.S. Pat. No. 10,640,773 issued onMay 5, 2020, which is a national stage application, filed under 35U.S.C. § 371, of International Application No. PCT/EP2014/058888, filedon Apr. 30, 2014, the content of which is incorporated herein byreference in its entirety and for all purposes.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

The content of the text file named “49839_505N01US_SL.txt,” which wascreated on Oct. 27, 2016 and is 2.16 KB in size, is hereby incorporatedherein by reference in its entirety.

The present invention relates to a plant sRNA extract for use as animmunosuppressive agent. The present invention further relates to plantmiRNA for use as an immunosuppressive agent.

In a further embodiment, the present invention relates to plant miRNAfor use as an anti-inflammatory agent.

A wide variety of eukaryotic organisms, including plants, animals, andfungi, have evolved several RNA-silencing pathways to protect theircells and genomes against invading nucleic acids, such as viruses ortransposons, and to regulate gene expression during development or inresponse to external stimuli (for review, see Baulcombe (2005) TrendsBiochem. Sci. 30: 290-293; Meins et al. (2005) Annu. Rev. Cell Dev.Biol. 21:297-318).

The discovery of microRNAs (miRNAs) is one of the major scientificbreakthroughs in recent years. These small non-coding RNA are changingthe way of thinking about the development of the immune system andregulation of immune functions (for a review see Bi Y. et al.;MicroRNAs: novel regulators during the immune response. J Cell Physiol.2009 March; 218(3):467-72; O'Connell R. M. et al.; Physiological andpathological roles for microRNAs in the immune system. Nat Rev Immunol.2010 February; 10(2):111-22). Endogenous miRNAs have been proposed astherapeutics or targets in the cure of cancer and other disorders (CalinG. A and Croce C M; MicroRNA signatures in human cancers. Nat RevCancer. 2006 November; 6(11):857-66).

Recently in addition to the known effects of intracellular orintraspecific miRNAs a more profound change of paradigm has beenproposed by the appearance of evidences that indicate the potential ofplant miRNAs to modulate human cells (Zhang L et al., Exogenous plantMIR168a specifically targets mammalian LDLRAP1: evidence ofcross-kingdom regulation by microRNA. Cell Research, 2012 22:107-126).In a recent highly debated report, Zhang et al (supra) discovered thatthe specific rice MIR168a could specifically target the mammalianlow-density lipoprotein receptor adapter protein, decreasing itsexpression in liver with consequence on LDL levels. If validated,diet-derived foreign microRNA absorption and function in consumingvertebrates would drastically alter our understanding of nutrition andecology. Therapeutic exploitation of RNAi in treating human disease isdifficult because these accessory processes are absent or diminished inmost animals. A recent report challenged multiple paradigms, suggestingthat ingested microRNAs (miRNAs) are transferred to blood, accumulate intissues, and exert canonical regulation of endogenous transcripts (seereview by Witwer and Hirsch (2014), Transfer and functional consequencesof dietary microRNAs in vertebrates: Concepts in search ofcorroboration, BioAssays).

In plants, RNA-silencing pathways have been shown to control a varietyof developmental processes including flowering time, leaf morphology,organ polarity, floral morphology, and root development (reviewed byMallory and Vaucheret (2006), Nat, Genet, 38:S31-36). All RNA-silencingsystems involve the processing of double-stranded RNA (dsRNA) into smallRNAs of 21 to 25 nucleotides (nt) by an RNaseIII-like enzyme, known asDicer or Dicer-like in plants (Bernstein et al. (2001) Nature 409:363-366; Xie et al. (2004) PLoS Biol 2, E104:0642-0652; Xie et al.(2005) Proc Natl Acad Sci USA 102: 12984-12989; Dunoyer et al., (2005)Nat Genet 37: 1356-1360). These small RNAs are incorporated intosilencing effector complexes containing an Argonaute protein (forreview, see Meister and Tuschl (2004) Nature 431: 343-349).

There are a number of major differences between plant and animal miRNA:Plant microRNA precursors are transcribed by RNA polymerase II as inanimals. However, in contrast to animals the entire process of plantmicroRNA biogenesis is undertaken within the plant nucleus. The maturemicroRNAs are exported out of the nucleus by Hasty, an exportin 5-likeprotein found in plants. There are fundamental differences between plantand animal microRNAs (Axtell, M J., et al. Vive la difference:biogenesis and evolution of microRNAs in plants and animals. GenomeBiology. 12(2011): p. 221-234.):

-   -   a) In animals miRNA precursors are sequentially processed by        Drosha followed by Dicer to produce miRNA duplexes whereas only        one Dicer-like enzyme (DCL1) is required in plants.    -   b) In plants miRNA duplexes are methylated at the 2′ OH group at        the 3′ termini.    -   c) The target recognition process between plants and animals is        different. In plants miRNAs direct mRNA cleavage of a microRNA        target whereas in animals miRNAs usually inhibit translation of        their targets.    -   d) In contrast to the nearly perfect match of plant miRNAs to        the exons of their target, hybridization of microRNA to targets        in animals is less stringent and only a canonical 7-8 nucleotide        “seed sequence” is known to be specific for microRNA target        recognition.

Studies focus on the role of plant miRNAs in plant; e.g. by thedevelopment of artificial miRNAs. Artificial microRNAs (amiRNAs) havebeen described in Arabidopsis targeting viral mRNA sequences (Niu et al.(2006) Nature Biotechnology 24: 1420-1428) or endogenous genes (Schwabet al. (2006) Plant Cell 18: 1121-1133). The amiRNA construct can beexpressed under different promoters in order to change the spatialpattern of silencing (Schwab et al. (2006) Plant Cell 18: 1121-1133).Artificial miRNAs replace the microRNA and its complementary starsequence in a miRNA precursor backbone and substitute sequences thattarget an mRNA to be silenced.

Silencing by endogenous miRNAs can be found in a variety of spatial,temporal, and developmental expression patterns (Parizotto et al. (2007)Genes Dev 18:2237-2242; Alvarez et al. (2006) Plant Cell 18: 1134-51).

As plant miRNAs are known to target plant target sequences, the effectof plant miRNAs in animals is unknown.

Immune-related and inflammatory diseases are the manifestation orconsequence of fairly complex, often multiple interconnected biologicalpathways which in normal physiology are critical to respond to insult orinjury, initiate repair from insult or injury, and mount innate andacquired defense against foreign organisms. Disease or pathology occurswhen these normal physiological pathways cause additional insult orinjury either as directly related to the intensity of the response, as aconsequence of abnormal regulation or excessive stimulation, as areaction to self, or as a combination of these.

Though the genesis of these diseases often involves multi-step pathwaysand often multiple different biological systems/pathways, interventionat critical points in one or more of these pathways can have anameliorative or therapeutic effect. Therapeutic intervention can occurby either antagonism of a detrimental process/pathway or stimulation ofa beneficial process/pathway.

Many immune related diseases are known and have been extensivelystudied. Such diseases include immune-mediated inflammatory diseasessuch as rheumatoid arthritis, immune mediated renal disease,hepatobiliary diseases, inflammatory bowel disease (IBD), psoriasis, andasthma, and non-immune-mediated inflammatory diseases, infectiousdiseases, immunodeficiency diseases, neoplasia, etc. However, there isstill a need for treatments of inflammatory diseases, which are moreefficient and/or exhibit fewer side effects than known treatments. Thesame applies for methods of preventing such diseases.

siRNAs have been proposed as a potential break-through in therapy ofcancer and other human diseases (Schiffelers R M 2004 NAR). The mainlimiting step to this therapeutic application is that canonical siRNAduplexes are potent activators of the mammalian innate immune system(Robbins et al., Oligonucleotides 2009) and therefore show undesiredoff-effects such as their induction of pro-inflammatory processes(Homung V et al, Nat Med 2005; Judge A D et al Nat Biotechnol 2005;Sloud M, J Mol Biol, 2005).

Other recent discoveries have shown that both high molecular weightdsRNA and siRNA induce inflammation triggering Toll-like receptorsignalling (Tatematsu M, Seya T, Matsumoto M. (2014). Beyond dsRNA:Toll-like receptor 3 signalling in RNA-induced immune responses. BiochemJ., 1; 458(2):195-201; Homung V et al, Nat Med 2005; Judge A D et al NatBiotechnol 2005; Sloud M, J Mol Biol, 2005).

Dendritic cells are the best known integrators of external signals, theyinsert their dendrites within epithelial cells in Peyer's patches, justbefore the ileo-caecal valve, to sense microorganisms (Swiatczak B,Rescigno M. How the interplay between antigen presenting cells andmicrobiota tunes host immune responses in the gut. Semin Immunol. 2012February; 24(1):43-9). dsRNAs and potentially miRNA are likelyrecognized as a viral threat and therefore would be expected to elicit apro-inflammatory response rather than the surprising anti-inflammatoryresponse that we report.

Therefore, plant miRNAs targeting beneficial gene regulatory processthrough specified food intake (i.e probiotics, specific diet regime)will likely be sensed by DCs trough specific mechanisms from the samereceptors but in a different manner than viral and human miRNAs (Croceet al., 2012 PNAS) Also, DC master regulators of immune responsemodulated inflammation and tolerance by interacting with T cellsdirectly or through cytokine production.

Human internally produced miRNAs are normally implicated in establishingand maintaining the cell fate of immune cells (e.g. miR-155 (O'ConnellR. M. et al; MicroRNA-155 promotes autoimmune inflammation by enhancinginflammatory T cell development. Immunity. 2010), miR-181a (Li Q J etal, 2007. miR-181a is an intrinsic modulator of T cell sensitivity andselection. Cell 129:147-161), miR-150 (Xiao C et al. 2007. MiR-150controls B cell differentiation by targeting the transcription factorc-Myb. Cell 131:146-159), miR-223 (Johnnidis J B et al., 2008.Regulation of progenitor cell proliferation and granulocyte function bymicroRNA-223. Nature 451:1125-1129) and miR146 (Taganov K D et al.,NF-kappaB-dependent induction of microRNA miR-146, an inhibitor targetedto signaling proteins of innate immune responses. Proc Natl Acad Sci USA2006; 103:12481-6.).

Thus, in one embodiment, the present invention relates a plant sRNAextract for use as an immunosuppressive agent.

Surprisingly, it was found that plant sRNA extract and plant miRNAsexhibit an anti-inflammatory effect, as opposed to what was observed forcanonical siRNAs and exogenously supplied mammalian miRNAs (Croce et al.2012 PNAS). Surprisingly, it could be shown that administering plantsRNA extract and/or plant miRNA in conjunction with differentinflammatory agents, dampens inflammation per se and also preventsinflammation.

Therefore, the experiments surprisingly show that plant sRNA extractsand plant miRNA are effective as anti-inflammatory agents, which canattenuate T cell proliferation and production both of inflammatorycytokines and co-stimulatory molecules in stimulated human dendriticcells, as described in the Examples.

This result shows a profound functional difference of plant miRNA ascompared to human miRNA, i.e an opposite role in immuno-modulatoryproperties. Therefore, plant sRNA extracts and plant miRNA exhibitunique, unexpected properties.

According to the present invention, a “plant sRNA extract” is understoodas a composition comprising plant material of a given plant varietywherein the amount of sRNA of said plant variety in comparison to otherRNA of the same plant variety in said composition is higher than innaturally occurring plant material of said plant variety.

In a preferred embodiment, the “plant sRNA extract” is understood as acomposition comprising plant material of a given plant variety whereinthe amount of sRNA of said plant variety in comparison to other RNA ofthe same plant variety in said composition is at least 10% higher thanin naturally occurring plant material of said plant variety, morepreferably at least 20%, 50%, 100% or 200% higher than in naturallyoccurring plant material of said plant variety.

Typically, the amount is determined as (w/v) or (w/w).

In more preferred embodiment, the “plant sRNA extract” according to thepresent application refers to the purified small RNA fraction of a plantoptionally dissolved in a suitable solvent. The plant miRNA extract whendissolved in a suitable solvent comprises less than 20% (w/v),preferably less than 10% (w/v), more preferably less than 5% (w/v), evenmore preferably less than less than 1% (w/v) of other plant components,in particular other RNA. A suitable solvent is preferably an aqueoussolution, which is more preferably a buffered aqueous solution, whichmay comprise further substance like RNAse inhibitors.

In case the extract is not dissolved, the extract may be solid, forexample it may be a dry, dried, or freeze-dried extract. Also, theextract may be in the form of a liquid or frozen fluid, such as asolution, suspension, dispersion, or the extract may be in the form of agel.

For extraction from plant, a complete plant or parts thereof of may beused. Preferably, plant sRNA may be extracted from leaves, flowers,roots, fruits, seeds or parts thereof. In particular strawberry fruitsmay be used.

The plant or parts thereof are preferably ground or shredded prior toextraction. For example, the plant or parts thereof may be pulverized,in particular in liquid nitrogen as described in Example 1.

In another preferred embodiment, the “plant sRNA extract” according tothe present application refers to the purified small RNA fraction of aplant optionally dissolved in a suitable solvent. The plant miRNAextract when dissolved in a suitable solvent comprises less than 20%(w/v), preferably less than 10% (w/v), more preferably less than 5%(w/v), even more preferably less than less than 1% (w/v) of other plantcomponents.

According to the present invention, the term “plant” encompasses anymember of the plant-kingdom according to the Linnaeus definition,encompassing both vascular and non-vascular plants.

In a preferred embodiment of the extract for use of the presentinvention, the sRNA has a length of 200 nucleotides or less, preferablya length of 120 nucleotides or less, preferably a length between 10 and120 nucleotides.

Therefore, in a preferred embodiment, the “sRNA plant extract” comprisesthe sRNA of a plant which has a length of 200 nucleotides or less,preferably a length of 120 nucleotides or less, preferably a lengthbetween 10 and 120 nucleotides.

In an even more preferred embodiment, the sRNA has a length of 10nucleotides or more.

Therefore, in a preferred embodiment, the “sRNA plant extract” comprisesthe sRNA of a plant which has a length of 10 nucleotides or more.

Thus, in an even more preferred embodiment of the present invention, thesRNA has a length of between 10 and 200 nucleotides, preferably a lengthof between 10 and 160 nucleotides, more preferably a length of between10 and 120 nucleotides, even more preferably a length of between 10 and100 nucleotides, even more preferably a length of between 10 and 50nucleotides, most preferably a length of between 10 and 25 nucleotides.

Therefore, in yet a further preferred embodiment, the “sRNA plantextract” comprises the sRNA of a plant which has a length of between 10and 200 nucleotides, preferably a length of between 10 and 160nucleotides, more preferably a length of between 10 and 120 nucleotides,even more preferably a length of between 10 and 100 nucleotides, evenmore preferably a length of between 10 and 50 nucleotides, mostpreferably a length of between 10 and 25 nucleotides.

The sRNA fraction extracted from plants containing the sRNA of a plantwhich has a length of between 10 and 25 nucleotides corresponds to themiRNA fraction. Therefore, this sRNA plant extract is particularlypreferred.

“sRNA” according to the present invention is small RNA, in particularRNA of a length of 200 nucleotides or less.

In a further preferred embodiment, the sRNA does not contain ribosomalRNA, or contains less than 70%, 50%, 10%, 10% or 1% ribosomal RNA (rRNA)of the same plant variety. For example, the ribosomal RNA is 5S rRNA,5.8S rRNA, 18S rRNA, or 28S rRNA.

Therefore, in a yet further preferred embodiment, the sRNA has a lengthof length of 120 nucleotides or less, preferably a length between 10 and120 nucleotides.

In a preferred embodiment of the extract for use of the invention, thesRNA is miRNA.

In a further embodiment, the present invention relates to miRNA for useas an immunosuppressive agent.

In a further embodiment, the present invention relates to a plant sRNAextract as defined above or plant miRNA for use in a method for treatingand/or preventing an inflammatory disease.

As used herein the term “inflammatory disease” and/or “inflammation”,used interchangeably, includes inflammatory abnormalities characterizedby enhanced immune response to harmful stimuli, such as pathogens,damaged cells, or irritants. Inflammatory diseases underlie a vastvariety of human diseases.

In a preferred embodiment, the inflammatory disease is selected from thegroup consisting of chronic prostatitis, Glomerulonephritis,Hypersensitivities, Pelvic inflammatory disease, Reperfusion injury,Sarcoidosis, Vasculitis, Interstitial cystitis, normocomplementemicurticarial vasculitis, pericarditis, myositis, anti-synthetase syndrome,scleritis, macrophage activation syndrome, Bechet's Syndrome, PAPASyndrome, Blau's Syndrome, gout, adult and juvenile Still's disease,cryropyrinopathy, Muckle-Wells syndrome, familial cold-inducedauto-inflammatory syndrome, neonatal onset multisystemic inflammatorydisease, familial Mediterranean fever, chronic infantile neurologic,cutaneous and articular syndrome, systemic juvenile idiopathicarthritis, Hyper IgD syndrome, Schnitzler's syndrome, TNFreceptor-associated periodic syndrome (TRAPSP), gingivitis,periodontitis, hepatitis, cirrhosis, pancreatitis, myocarditis,vasculitis, gastritis, gout, gouty arthritis, and inflammatory skindisorders, selected from the group consisting of psoriasis, atopicdermatitis, eczema, rosacea, urticaria, and acne.

In a further preferred embodiment, said inflammatory disease is anautoimmune disease.

In a particularly preferred embodiment, said autoimmune disease isselected from the group consisting of multiple sclerosis, rheumatoidarthritis; psoriatic arthritis, discoid lupus erythematosus, systemiclupus erythematosus (SLE); ulcerative colitis; Crohn's disease; benignlymphocytic angiitis, autoimmune lymphoproliferative syndrome,sarcoidosis, autoimmune thrombocytopenic purpura, idiopathicthrombocytopenic purpura, pure red cell aplasia, Sjogren's syndrome,rheumatic disease, polymyalgia rheumatica, mixed connective tissuedisease, inflammatory rheumatism, degenerative rheumatism,extra-articular rheumatism, juvenile arthritis, juvenile rheumatoidarthritis, systemic juvenile idiopathic arthritis, arthritis uratica,muscular rheumatism, chronic polyarthritis, reactive arthritis, Reiter'ssyndrome, rheumatic fever, relapsing polychondritis, Raynaud'sphenomenon, vasculitis, cryoglobulinemic vasculitis, ANCA-associatedvasculitis, temporal arteritis, giant cell arteritis, Takayasuarteritis, Behcet's disease, antiphospholipid syndrome, myastheniagravis, autoimmune haemolytic anemia, Guillain-Barre syndrome, chronicimmune polyneuropathy, chronic inflammatory demyelinatingpolyneuropathy, autoimmune thyroiditis, insulin dependent diabetesmellitus, type I diabetes, Addison's disease, membranousglomerulonephropathy, polyglandular autoimmune syndromes, Goodpasture'sdisease, autoimmune gastritis, autoimmune atrophic gastritis, perniciousanemia, Pemphigus, Pemphigus vulgaris, cirrhosis, primary biliarycirrhosis, idiopathic pulmonary fibrosis, myositis, dermatomyositis,juvenile dermatomyositis, polymyositis, fibromyositis, myogelosis,celiac disease, celiac sprue dermatitis, immunoglobulin A nephropathy,Henoch-Schonlein purpura, Evans syndrome, atopic dermatitis, psoriasis,psoriasis vulgaris, psoriasis arthropathica, Graves' disease, Graves'ophthalmopathy, scleroderma, systemic scleroderma, progressive systemicscleroderma, diffuse scleroderma, localized scleroderma, Crest syndrome,asthma, allergic asthma, allergy, primary biliary cirrhosis, Hashimoto'sthyroiditis, fibromyalgia, chronic fatigue and immune dysfunctionsyndrome (CFIDS), primary myxedema, sympathetic ophthalmia, autoimmuneinner ear disease, autoimmune uveitis, autoimmune chronic activehepatitis, collagen diseases, ankylosing spondylitis, periarthritishumeroscapularis, panarteritis nodosa, polyarteritis nodosa,chondrocalcinosis, Wegener's granulomatosis, microscopic polyangiitis,chronic urticaria, bullous skin disorders, pemphigoid, bullouspemphigoid, cicatricial pemphigoid, vitiligo, atopic eczema, eczema,chronic urticaria, autoimmune urticaria, normocomplementemic urticarialvasculitis, hypocomplementemic urticarial vasculitis, alopecia areata,alopecia universalis, alopecia totalis, Devic's disease, perniciousanemia, childhood autoimmune hemolytic anemia, idiopathic autoimmunehemolytic anemia, refractory or chronic Autoimmune Cytopenias,Prevention of development of Autoimmune Anti-Factor VIII Antibodies inAcquired Hemophilia A, Cold agglutinin disease, Neuromyelitis Optica,Stiff Person Syndrome, gingivitis, periodontitis, pancreatitis,myocarditis, gastritis, gout, gouty arthritis, idiopathic pericarditis,anti-synthetase syndrome, scleritis, macrophage activation syndrome,PAPA Syndrome, Blau's Syndrome, adult and juvenile Still's disease,cryopyrin associated periodic syndrome, Muckle-Wells syndrome, familialcold auto-inflammatory syndrome, neonatal onset multisystem inflammatorydisease, chronic infantile neurologic cutaneous and articular syndrome,familial Mediterranean fever, Hyper IgD syndrome, Schnitzler's syndrome,autoimmune retinopathy, age-related macular degeneration, and TNFreceptor-associated periodic syndrome (TRAPS).

In a yet further preferred embodiment, the inflammatory disease is anallergic disease.

In a particularly preferred embodiment, said allergic disease isselected from the group consisting of

-   a) allergic respiratory disease, such as bronchial asthma, pediatric    asthma, allergic asthma, atopic asthma, aspirin asthma, or allergic    bronchitis,-   b) allergic nasal disease, such as allergic rhinitis, vernal    catarrh, hay fever, or chronic allergic rhinitis,-   c) an allergic skin disease, such as atopic dermatitis,-   d) an allergic ocular disease, such as hay fever, seasonal allergic    conjunctivitis, or chronic allergic conjunctivitis,-   e) hypersensitivity pneumonitis,-   f) contact dermatitis, and-   g) food allergy.

In a further preferred embodiment, said inflammatory disease ispsoriasis.

In a further preferred embodiment, said inflammatory disease isinflammatory bowel disease, more preferably Crohn's disease orulcerative colitis.

In a further preferred embodiment, said inflammatory disease isulcerative colitis.

As used herein, “inflammatory bowel disease” also refers to a relateddisease and refers to all types and stages of inflammatory bowel disease(IBD), including, but not limited to: Crohn's disease and ulcerativecolitis (UC). Optionally, conditions relating to IBD include, e.g.,Collagenous colitis, Lymphocytic colitis, Ischaemic colitis, Diversioncolitis, Behcet's disease, Indeterminate colitis.

As used herein, “psoriasis” also refers to a related disease and refersto all types and stages of psoriasis, including, but not limited to:Nonpustular Psoriasis including Psoriasis vulgaris and Psoriaticerythroderma (erythrodermic psoriasis), Pustular psoriasis includingGeneralized pustular psoriasis (pustular psoriasis of von Zumbusch),Pustulosis palmaris et plantaris (persistent palmoplantar pustulosis,pustular psoriasis of the Barber type, pustular psoriasis of theextremities), Annular pustular psoriasis, Acrodermatitis continua,Impetigo herpetiformis. Optionally, conditions relating to psoriasisinclude, e.g., drug-induced psoriasis, Inverse psoriasis, Napkinpsoriasis, Seborrheic-like psoriasis, Guttate psoriasis, Nail psoriasis,Psoriatic arthritis.

In the Examples, it is shown that plant sRNA extracts and plant miRNAsattenuate T cell proliferation and the expression and/or production ofIL-1β, TNFα, CD80, CD86 and CD83 of stimulated dendritic cells.Therefore, plant sRNA extracts and plant miRNAs are in particular usefulin the treatment and/or prevention of autoimmune diseases orinflammatory diseases, in which IL-1β, TNFα, CD80, CD86 and/or CD83 playa role, and/or wherein dendritic cells are dysregulated. For example inpatients with Crohn's disease, pro-inflammatory Th17 and Th1 cytokinesoutweigh the effect of anti-inflammatory cytokines secreted byregulatory T cells (Treg). This results in an imbalance ofpro-inflammatory and anti-inflammatory cytokines. Thus, Crohn's diseasemay be treated and/or prevented with plant miRNA according to thepresent invention.

Thus, in a further preferred embodiment, the inflammatory disease ischaracterized by an increase in CD4⁺ T cell proliferation.

Thus, in a further preferred embodiment, the inflammatory disease ischaracterized by an increase in expression or production of at least oneof inflammatory biomarker, in particular selected from IL-1β and TNFαand/or wherein the inflammatory disease is characterized by adysregulation of the cytokines or co-receptors in at least, and/or by adysregulation of immune function as detectable in changing of DCactivity (i.e. costimulatory molecules expression).

Thus, in a further preferred embodiment, the inflammatory disease ischaracterized by an increase in expression or production of IL-10, suchas lymphoma, in particular non-Hodgkin's lymphoma, and Burkitt lymphoma,melanoma and non-small cell lung cancer.

A “patient” is understood as animal, in particular a human in need oftreatment and/or prevention. Accordingly, the plant sRNA extract orplant miRNA for use in prevention and/or treatment is suitable for theprevention and/or treatment of animals, in particular humans in need oftreatment and/or prevention.

As shown in the Examples, plant sRNA extracts from monocot and dicotplants are effective in attenuating the T cell proliferation.

Surprisingly, the total sRNA extract from strawberry, namely Fragariavesca, is even more effective.

Thus, the sRNA extract for use may be from any plant, in particular froma monocot or dicot. For example, plant sRNA extracted from soybean,maize, or strawberry may be used.

In a preferred embodiment of the plant sRNA extract or plant miRNA foruse of the invention, the plant is a flowering plant.

In a more preferred embodiment of the plant sRNA extract or plant miRNAfor use of the invention, the plant is a Rosacea spp.

Rosaceae plants are known to a skilled person in include the subfamilyRosoideae, in particular rose, blackberry, raspberry, strawberry,Potentilla and Geum; the subfamily Amygdaloideae, in particular pomefruits, traditionally known as subfamily Maloideae or Pyroideae, likeapple, cotoneaster, and hawthorn; the Spiraeoideae; and the subfamilyDryadoideae, in particular the genera Dryas, Cercocarpus, Chamaebatia,Cowania, and Purshia.

In an even more preferred embodiment of the plant sRNA extract or plantmiRNA for use of the invention, the plant is a strawberry, mostpreferred Fragaria vesca.

In a yet further preferred embodiment, one or more different plant sRNAextracts and/or plant miRNAs are administered. For example 2, 3, 4, 5,6, 7, 8, 9 or 10 different plant sRNA extracts may be administered. Forexample, sRNA extracts from strawberry and soybean may be administered.

The one or more different plant sRNA extracts may be administeredtogether or separately, or they may be administered simultaneously, orat different time points.

Moreover, some synthetic specific plant miRNAs are effective inattenuating T cell proliferation and the expression and/or production ofinflammatory cytokines IL-1β, TNFα, and immune reactivity of stimulateddendritic cells by means of CD80, CD86 and CD83 proper expression. Asynthetic miRNA is understood as a miRNA which exists naturally in aplant, which is obtained by chemical synthesis. Alternatively, thespecific plant miRNAs may be purified from plants. The synthesis ofmiRNA is known in the art and is for example described in Example 1.

The plant miRNA for use of the invention is preferably a purified miRNA.

A purified plant miRNA typically contains less than 10%, 1%, 0,01% or0,001% of other components, in particular plant components.

The one or more different plant miRNAs may be administered together orseparately, or they may be administered simultaneously, or at differenttime points.

Preferably, at least one plant sRNA extract and/or two or more purifiedmiRNAs are administered.

Surprisingly, miR168 from Fragaria vesca is highly effective regardingits immunesuppressive effect, as shown in the examples. Thus, in apreferred embodiment, the plant miRNA for use according to the inventionis F. vesca miR168.

In particular, plant sRNA extracts from one plant or more than one plantmay be administered. For example plant sRNA extracts from two differentplant varieties or different plant families may be administered.

“More than one” is understood to encompass for example 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 14, 20, 40 or more.

Plant sRNA extracts can be obtained as described in the Examples.

In a further preferred embodiment, the two or more plant sRNA extractsmay be administered as a mixture of plant sRNA extracts. In particular,plant sRNA extracts from two different plant varieties or differentplant families may be mixed and may be administered thereafter.

The plant sRNA extracts preferably contain methylated miRNAs or themiRNAs of the extract are methylated. In a further preferred embodiment,a plant miRNA for use is a methylated miRNA. A methylated miRNA for useaccording to the invention is preferably methylated at the 2′OH group ofthe 3′ terminus.

The miRNAs for use according to the invention may be producedrecombinantly using miRNA expression constructs, using methods known inthe art (Schwab, R., Ossowski, S., Riester, M., Warthmann, N., andWeigel, D. (2006). Highly specific gene silencing by artificialmicroRNAs in Arabidopsis. The Plant Cell 18, 1121-1133), or may beisolated from plants.

Preferably, mature miRNA molecules are administered.

A number of miRNAs are described in the prior art and may be usedaccording to the invention. These miRNAs are described in a large numberof plant species as shown in Table 1 below (Kozomara, A., andGriffiths-Jones, S. (2011). miRBase: integrating microRNA annotation anddeep-sequencing data. Nucleic Acids Research 39, D152-D157). ThesemiRNAs were also described in Fragaria ananassa (Ge, A., Shangguan, L.,Zhang, X., Dong, Q., Han, J., Liu, H., Wang, X., and Fang, J. (2012).Deep sequencing discovery of novel and conserved microRNAs in strawberry(Fragaria×ananassa). Physiologia Plantarum).

TABLE 1 miRNA Target sequence Cleaved Genes miR156/157 (12)SQUAMOSA-promoter binding SPL2, SPL3, SPL10 miR158 (2) Unknown miR159(6) MYB, TCP MYB33, MYB65, TCP2, TCP3, TCP4, TCP10, TCP24, miR160 (3)ARF ARF10, ARF17 miR161 (1) Pentatricopeptide repeat At1g06580 miR162(2) Dicer DCL1 miR163 (1) Methyl transferases miR164 (3) NAC-domaintranscription factor CUC1, CUC2, NAC1, At5g07680, At5g61430 miR165/166(9) HD-ZIP transcription factor PHB, PHV, REV, C3HDZIP1 miR167 (4) ARFARF8 miR168 (2) AGO AGO1 miR169 (14) CCAAT-binding factor HAP2-likeprotein At3g05690 miR170/171 (4) GRAS-domain transcription factorSCL6-III, SCL6-IV miR172 (5) APETALA2-like transcription factor AP2,TOE1, TOE2, TOE3 miR173 (1) Unknown miR393 (2) AFB3 Auxin receptorsTIR1, AFB1, AFB2, miR394 (2) F-box protein At1g27340 miR395 (6) ATPsulfurylase APS4 miR396 (2) Growth-regulating-factor transcriptionfactor GRL1, GRL2, GRL3, Rhodenase-like protein, Kinesin-like proteinGRL7, GRL8, GRL9 miR397 (2) Laccase, Beta-6 tubulin At2g29130,At2g38080, At5g60020 miR398 (3) CSD, Cytochrome C oxidase subunit VCSD1, CSD2, At3g15640 miR399 (6) Phosphate transporter

Preferably, miR156, miR168, osamiR168, or mixtures thereof are used.

A particularly preferred miRNA is miR168, in particular Fragaria vescamiR168.

A “miRNA” or “microRNA” for use according to the present invention isunderstood as oligoribonucleic acid, of about 19 to 24 nucleotides (nt)in length, which regulates expression of a polynucleotide comprising atarget sequence. miRNAs are non-protein-coding RNAs and have beenidentified in both animals and plants. miRNAs are derived in plants viaDicer-like 1 processing of larger precursor polynucleotides. The term“miRNA” also encompasses “artificial miRNA”, which comprise a miRNAsequence that is synthetically designed to silence a target sequence. Inparticular, such artificial miRNA exhibits 80% or more, preferably 90%or more, even more preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity with the nucleotide sequence of theunmodified miRNA or miRNA precursor backbone. In a preferred embodiment,the plant miRNA is methylated at the 2′OH group at the 3′ terminus.

In a preferred embodiment, endogenous miRNAs are used, as the mechanismappears to be target-independent.

“miRNA precursor backbone” is a polynucleotide that provides thebackbone structure necessary to form a hairpin RNA structure whichallows for the processing and ultimate formation of the miRNA.Preferably, the backbone is from strawberry, rice, maize or soybean.microRNA precursor backbones have been described previously. Forexample, US20090155910A1 (WO 2009/079532) discloses the followingsoybean miRNA precursor backbones: 156c, 159, 166b, 168c, 396b and 398b,and US 20090155909A1(WO 2009/079548) discloses the following maize miRNAprecursor backbones: 159c, 164h, 168a, 169r, and 396h. Examples of miRNAprecursor backbones include the miRNA GM-396b precursor backbone oractive variants thereof and the miRNA GM-159 precursor backbone oractive variants thereof, as described in WO 2012/154824.

A “miRNA expression construct” is understood as DNA construct whichcomprises a miRNA precursor backbone having a polynucleotide sequenceencoding a miRNA and a star sequence. Such miRNA expression constructmay be in a suitable vector, to allow expression in a suitable host,followed by isolation of the miRNA. Such expression systems are known toa skilled person and are for example described in (Schwab, R., Ossowski,S., Riester, M., Warthmann, N., and Weigel, D. (2006). Highly specificgene silencing by artificial microRNAs in Arabidopsis. The Plant Cell18, 1121-1133)

The plant miRNAs and plant sRNA extracts for use surprisingly exhibitanti-inflammatory activity. Such effect may be enhanced by combining theplant miRNAs or and plant sRNA extracts with other knownanti-inflammatory agents.

Thus, in a further preferred embodiment, the plant sRNA extract or theplant miRNA for use of the invention is administered in combination withat least one other anti-inflammatory agent.

In a more preferred embodiment, said other anti-inflammatory agent isselected from the group consisting of a corticosteroid, a statin,interferon beta, a nonsteroidal anti-inflammatory drug (NSAID),methotrexate, Cyclosporin A and a disease-modifying anti-rheumatic drug(DMARD).

In another embodiment, the plant sRNA extract or the plant miRNA for useaccording to the invention is comprised in a pharmaceutical composition.

Thus, in a further embodiment, the present invention relates to apharmaceutical composition comprising at least one plant miRNA or plantsRNA extract as defined herein, for use in a method for treating and/orpreventing an inflammatory disease, as defined above.

In a preferred embodiment, the pharmaceutical composition comprises atleast one plant miRNA or plant sRNA extract and at least one carrier.Carriers are generally known to those skilled in the art and includesaline, sugars, polypeptides, polymers, lipids, creams, gels, micellematerials, and metal nanoparticles. In one more preferred embodiment,the carrier comprises at least one of the following: water, a glucosesolution, a polycationic binding agent, a cationic lipid, a cationicmicelle, a cationic polypeptide, a hydrophilic polymer grafted polymer,a non-natural cationic polymer, a cationic polyacetal, a hydrophilicpolymer grafted polyacetal, a liposome, a ligand functionalized cationicpolymer, a ligand functionalized-hydrophilic polymer grafted polymer,and a ligand functionalized liposome. In another more preferredembodiment, the polymers comprise a biodegradable histidine-lysinepolymer, a biodegradable polyester, such as poly(lactic acid) (PLA),poly(glycolic acid) (PGA), and poly(lactic-co-glycolic acid) (PLGA), apolyamidoamine (PAMAM) dendrimer, a cationic lipid, or a PEGylated PELCationic lipids include DOTAP, DOPE, DC-Chol/DOPE, DOTMA, andDOTMA/DOPE. In a yet further preferred embodiment, exosomes may be used.Such exosomes and their preparation are described e.g. in Montecalvo etal. (2012, Blood, 119: 756-766, and Stoorvogel, 2012, Blood, 119:646-648). Further suitable pharmaceutical carriers are e.g. described inRemington's Pharmaceutical Sciences, supra, a standard reference text inthis field. The pharmaceutical composition may further compriseconventional pharmaceutical additives and adjuvants, excipients ordiluents, including, but not limited to, water, gelatin of any origin,vegetable gums, ligninsulfonate, talc, sugars, starch, gum arabic,vegetable oils, polyalkylene glycols, flavoring agents, preservatives,stabilizers, emulsifying agents, buffers, lubricants, colorants, wettingagents, fillers, and the like.

A particularly preferred carrier is DOTAP, as used in the Examples.

Preferably, the pharmaceutical composition comprises at least onepharmaceutically acceptable RNAse inhibitor.

In still another embodiment, the carrier is a polymer, e.g. ahistidine-lysine copolymer that forms a nanoparticle with the miRNAmolecule(s). The diameter of the nanoparticle is about typically 100 nmto about 1000 nm.

The invention thus also provides a nanoparticle comprising one or moreof the plant miRNA or plant sRNA extract for use of the invention. In anembodiment, the nanoparticle further comprises a carrier, such as one ormore of those described herein, and optionally a targeting ligand.

In one further embodiment of the present invention, said plant miRNA orplant sRNA extract is comprised in a food additive or dietarysupplement. Preferably, the dietary supplement, food additive, food orfoodstuff comprises at least one RNAse inhibitor which is acceptable forsuch compositions. In particular, such RNAse inhibitor is not toxic.

Methods for determining the amount of sRNA or specific miRNA are knownin the art. For example chip-based detection assays, like Agilent smallRNA assays, or Millipore SmartRNAplex™ miRNA profiling assay, orPCR-based detection assays, like Qiagen miScript™ PCR System, TaqMan®MicroRNA Assays, or Exiqon miRCURY LNA™ Detection may be used.

Such food additive may be added to a food product, foodstuff, dietarysupplement, nutritional supplement or a supplement composition for afood product or a foodstuff, for example beverages (e.g. but not limitedto sports beverages, functional waters, juices, smoothies; instantdrinks), soups, dairy products (e.g. but not limited to single shotyoghurt drinks), nutritional bars, and spreads, in particular beveragesand nutritional bars.

For example, a Fragaria vesca sRNA extract may be prepared in liquidform such as an aqueous solution which optionally contains at least oneRNAse inhibitor. Such solution can be used as food additive e.g. forbeverages or dairy products. As used herein, the term food productrefers to any food or feed suitable for consumption by humans oranimals. The food product may be a prepared and packaged food (e.g.,mayonnaise, salad dressing, bread, or cheese food) or an animal feed(e.g., extruded and pelleted animal feed, coarse mixed feed or pet foodcomposition). As used herein, the term foodstuff refers to any substancefit for human or animal consumption. The term dietary supplement refersto a small amount of a compound for supplementation of a human or animaldiet packaged in single or multiple dose units. Dietary supplements donot generally provide significant amounts of calories but may containother micronutrients (e.g., vitamins or minerals). The term nutritionalsupplement refers to a composition comprising a dietary supplement incombination with a source of calories.

Food products or foodstuffs are for example beverages such asnon-alcoholic and alcoholic drinks as well as liquid preparation to beadded to drinking water and liquid food, non-alcoholic drinks are forinstance soft drinks, sport drinks, fruit juices, such as for exampleorange juice, apple juice and grapefruit juice; lemonades, teas,near-water drinks and milk and other dairy drinks such as for exampleyoghurt drinks, and diet drinks. In another embodiment food products orfoodstuffs refer to solid or semi-solid foods comprising the compositionaccording to the invention. These forms can include, but are not limitedto baked goods such as cakes and cookies, puddings, dairy products,confections, snack foods, or frozen confections or novelties (e.g., icecream, milk shakes), prepared frozen meals, candy, snack products (e.g.,chips), liquid food such as soups, spreads, sauces, salad dressings,prepared meat products, cheese, yoghurt and any other fat or oilcontaining foods, and food ingredients (e.g., wheat flour). The termfood products or foodstuffs also includes functional foods and preparedfood products, the latter referring to any pre-packaged food approvedfor human consumption.

The food products or foodstuffs may already contain RNA prior to theaddition of a food additive comprising a plant miRNA or plant sRNAextract for use according to the invention. This may be the case forfood products or foodstuff being, containing or derived from plants orparts thereof, such as fruits, or fruit juices.

In this embodiment, the resulting food products or foodstuffs contain anenlarged amount and/or concentration of plant miRNA or plant sRNAextract compared to the food products or foodstuffs without addition ofsuch food additive.

In a preferred embodiment, such food products or foodstuffs contain anamount of plant sRNA which is at least 10% higher than the amount in thefood product or foodstuff without addition of such food additivecomprising a plant sRNA extract, more preferably the amount is at least20%, 50%, 100% or 200% higher than in the food product or foodstuffwithout addition of such food additive comprising a plant sRNA extract.

In a further preferred embodiment, such food products or foodstuffscontain an amount of total plant miRNA which is at least 10% higher thanthe amount in the food product or foodstuff without addition of suchfood additive containing one or more specific miRNAs, more preferablythe amount is at least 20%, 50%, 100% or 200% higher than in the foodproduct or foodstuff without addition of such containing one or morespecific miRNAs.

In a yet further preferred embodiment, such food products or foodstuffscontain an amount of a specific plant miRNA which is at least 10% higherthan the amount of the specific plant miRNA in the food product orfoodstuff without addition of such food additive containing one or morespecific miRNAs, more preferably the amount is at least 20%, 50%, 100%or 200% higher than in the food product or foodstuff without addition ofsuch containing one or more specific miRNAs.

Animal feed including pet food compositions advantageously include foodintended to supply necessary dietary requirements, as well as treats(e.g., dog biscuits) or other food supplements. The animal feedcomprising the composition according to the invention may be in the formof a dry composition (for example, kibble), semi-moist composition, wetcomposition, or any mixture thereof. Alternatively or additionally, theanimal feed is a supplement, such as a gravy, drinking water, yoghurt,powder, suspension, chew, treat (e.g., biscuits) or any other deliveryform.

Food compositions are preferably administered orally. Dietarysupplements are preferably administered orally.

Therefore, in a yet further embodiment, the present invention relates toa food product comprising a plant sRNA extract or a plant miRNA asdefined above.

Therefore, in a yet further embodiment, the present invention relates toa dietary supplement comprising a plant sRNA extract or a plant miRNA asdefined above.

In a yet further embodiment, the present invention relates to a methodfor producing a second food product, comprising adding to a first foodproduct a plant sRNA extract, or a plant miRNA as defined herein.

For example, a plant sRNA extract as defined above may be added to abeverage and may be mixed, resulting in a beverage containing orenriched with plant sRNA extract. In case the first food product doesnot contain plant sRNA, the second food product contains plant sRNA.

Therefore, the first food product is in a preferred embodiment a foodproduct which does not contain plant sRNA and/or plant miRNA.

In another preferred embodiment, the first food product contains plantsRNA and/or plant miRNA. In one preferred embodiment, such second foodproduct contain an amount of plant sRNA which is at least 10% higherthan the amount in the first food product, more preferably the amount isat least 20%, 50%, 100% or 200% higher than in first food product.

In a further preferred embodiment, such second food product contains anamount of total plant miRNA which is at least 10% higher than the amountin the first food product, more preferably the amount is at least 20%,50%, 100% or 200% higher than in the first food product.

In a yet further preferred embodiment, such second food product containsan amount of a specific plant miRNA which is at least 10% higher thanthe amount in the first food product, more preferably the amount is atleast 20%, 50%, 100% or 200% higher than in the first food product.

Dietary supplements may be delivered in any suitable format. Inpreferred embodiments, dietary supplements are formulated for oraldelivery. The ingredients of the dietary supplement of this inventionare contained in acceptable excipients and/or carriers for oralconsumption. The actual form of the carrier, and thus, the dietarysupplement itself, is not critical. The carrier may be a liquid, gel,gelcap, capsule, powder, solid tablet (coated or non-coated), tea, orthe like.

In other embodiments, the dietary supplement is provided as a powder orliquid suitable for adding by the consumer to a food or beverage. Forexample, in some embodiments, the dietary supplement can be administeredto an individual in the form of a powder, for instance to be used bymixing into a beverage, or by stirring into a semi-solid food such as apudding, topping, sauce, puree, cooked cereal, or salad dressing, forinstance, or by otherwise adding to a food e.g. enclosed in caps of foodor beverage container for release immediately before consumption.

The dosage of miRNA for use according to the invention as food additive,of course, vary depending upon known factors, such as its mode and routeof administration; the age, health and weight of the recipient; thenature and extent of the symptoms; the kind of concurrent treatment; thefrequency of treatment; and the effect desired which can be determinedby the expert in the field with normal trials, or with the usualconsiderations regarding the formulation of a miRNA.

The dosage of miRNA for use according to the invention in apharmaceutical composition can be determined by the expert in the fieldwith normal preclinical and clinical trials, or with the usualconsiderations regarding the formulation of pharmaceutical composition.

The pharmaceutical compositions may be administered as single dose ormultiple doses.

The compositions according to the present invention may be in anygalenic form that is suitable for administering to the animal bodyincluding the human body, more in particular in any form that isconventional for oral administration, e.g. in solid form, for exampletablets, pills, granules, dragees, capsules, and effervescentformulations such as powders and tablets, or in liquid form, forinstance in the form of solutions, emulsions or suspensions, for exampleas pastes and oily suspensions. The pastes may be filled into hard orsoft shell capsules, whereby the capsules feature e.g. a matrix of(fish, swine, poultry, cow) gelatin, plant proteins or lignin sulfonate.Examples for other application forms are forms for transdermal,parenteral, topical or injectable administration. The pharmaceuticalcompositions may be in the form of controlled (delayed) releaseformulations.

In a preferred embodiment the compositions according to the inventionare in the form of a tablet, a pill, a granule, a dragee, a capsule oran effervescent formulation.

In a yet further preferred embodiment, the present invention relates toa method of treating or preventing an inflammatory disease, comprisingadministering to a patient in need thereof a pharmaceutically effectiveamount of plant sRNA extract or plant miRNA.

In a yet further preferred embodiment, the present invention relates toa method of preventing an inflammatory disease, comprising administeringto a human a plant sRNA extract or plant miRNA, which is preferablycomprised in a dietary supplement, food or food additive. The human isin one preferred embodiment a patient in need thereof. In anotherembodiment the human is a healthy person, in particular a person notsuffering from an inflammatory disease.

The dietary supplement may be administered as single dose or multipledoses.

In a yet further embodiment of the food product of the present inventionor the method of method for producing a second food product, the miRNAis F. vesca miR168.

FIGURES LEGEND

FIGS. 1A-1H: Examples of the sRNA profiles obtained according toExample 1. FIG. 1A. Ladder profile showing the six RNA markers innucleotides (nt); FIG. 1B. Profile obtained by running a total RNAsample encompassing the high molecular weight fraction, identifiable bythe 28S and 18S peaks, and the sRNA fraction of low molecular weight, onthe left of the electropherogram. FIGS. 1C-1G. Examples of the sRNAfractions used in the experiments here presented. FIG. 1H.Electropherogram of a sample that has been excluded after analysis sincehigh molecular weight RNA contamination (18S and 28S) is present.

FIG. 2: shows the sequences of duplex structures of Fragaria vescaFvmiR156 (A) FvmiR168 (B), Oryza sativa OsamiR168 (C) and a sGFP (D).me: methyl group.

FIG. 3: Effects of different plant sRNA extracts on T cellproliferation. MLR results are shown. Any treatment is statisticallysignificant with respect to the control (DC+T w/o sRNA treatment)

FIG. 4: shows the Experimental design to obtain dendritic cells formiRNA treatment and to evaluate the effect of plant miRNA on stimulatedDC.

FIG. 5: Effects of plant miRNA treatment in DC immune function. Effectsof miRNA treatment on IL-1β, TNFα, IL-10 and IL-6 production. Mean+SD,N=5, *p<0.05, **p<0.01 T test, p<0.05, Kruskal Wallis test, treatment vsno pretreatment

FIG. 6: Effects of plant miRNA treatment in DC immune function. Effectsof miRNA treatment on costimulatory molecules expression. Mean+SD, N=3,*p<0.05, **p<0.01 T test, p<0.05, Kruskal Wallis test, treatment vs nopretreatment

FIG. 7: Effects of endogenous human miRNA treatment in DC immunefunction. Effects miRNA treatment on IL-1β, TNFα, IL-10 production.Mean+SD, N=5, *p<0.05, **p<0.01 T test, p<0.05, Kruskal Wallis test,treatment vs no pretreatment

FIG. 8: Effects of endogenous human miRNA treatment in DC immunefunction. Effects of miRNA treatment on costimulatory moleculesexpression. Mean+SD, N=5, *p<0.05, **p<0.01 T test, p<0.05, KruskalWallis test, treatment vs no pretreatment

FIG. 9: Ability of T cells to produce INFγ in response to LPS-loaded DCspreviously exposed to different plant and human miRNAs as well as totalFvsRNA fraction. Mean+SD, N=5, *p<0.05, **p<0.01 T test, § p<0.05,Kruskal Wallis test, treatment vs no pretreatment.

FIG. 10: Effects of miRNA treatment on T cell proliferation. MLR resultsafter treatment with different plant and human miRNA as well as totalFvRNA fraction are shown. Mean+SD, N=4, *p<0.05, **p<0.01 T test,treatment vs not pretreatment MLR assay showed that FvmiR168 and FvsRNAmodulate T cell proliferation.

SEQUENCES

corresponds to the sequence of methylated FvmiR156 SEQ ID No. 1(5′-UUGACAGAAGAGAGUGAGCACmeth-3′). corresponds to the sequence ofmethylated FvmiR156* SEQ ID No. 2 (5′-AGCUCUUUCUCUUUCUGUCACUmeth-3′).corresponds to the sequence of methylated FvmiR168. SEQ ID No. 3(5′-UCGCUUGGUGCAGGUCGGGAAmeth-3′) corresponds to the sequence ofmethylated FvmiR168* SEQ ID No. 4 (5′-CCCGCCUUGCAUCAACUGAAUmeth-3′).corresponds to the sequence of methylated osamiR168. SEQ ID No. 5(5′-UCGCUUGGUGCAGAUCGGGACmeth-3′). corresponds to the sequence ofmethylated osamiR168* SEQ ID No. 6 (5′-GAUCCCGCCUUGCACCAAGUGAAUmeth-3′).corresponds to the sequence of small duplex sGFP. SEQ ID No. 7(5′-AGAACGGCAUCAAAGCCAACU-3′ (sGFP)).corresponds to the sequence of the  second strand of small duplexSEQ ID No. 8 sGFP (3′-UUGGCUUUGAUGCCGUUCUUUU-5′ (as GFP)).

EXAMPLES Example 1: Preparation of a Plant sRNA Extracts and Synthesisof miRNAs miR156, miR168, osamiR168 and Small Duplex sGFP (FIGS. 1A-1Hand 2)

Plant sRNAs were extracted using the mirPremier microRNA Isolation Kit(Sigma-Aldrich) according to the manufacturer's protocol. We collectedseveral plant materials, encompassing the plant kingdom: strawberryfruits from Fragaria vesca (cv. Hawaii 4), blueberry, raspberry fruits,apple skin, cabbage leaves and flowers; Traminer grape; fern leaves;Arabidopsis thaliana leaves; wild rice and corn. All of them werepulverized in liquid nitrogen. One hundred milligrams were mixed withthe buffer provided in the kit. The small RNA fraction was separatedfrom the high molecular weight RNA by the use of columns included in thekit. Small RNA was finally eluted using RNAse-free water.

After each extraction, samples will be analyzed using the Agilent RNA6000 Nano Kit through the BioAnalyzer instrument. Examples of the sRNAprofiles used in the patent are showed in FIGS. 1A-1H. FIG. 1A shows thesix RNA markers in nucleotides (nt); FIG. 1B shows the profile obtainedby running a total RNA samples encompassing the high molecular weightfraction, identifiable by the 28S and 18S peaks, and the sRNA fractionof low molecular weight, on the left of the electropherogram. FIGS.1C-1G panels show examples of the sRNA fractions used in the experimentshere presented. FIG. 1H shows a sample that has been excluded after theanalysis since high molecular weight RNA contamination (18S and 28S) ispresent. Furthermore we analyzed each sRNA preparation using the AgilentSmall RNA kit. Through BioAnalyzer analysis we characterized the size ofthe sRNA present in each fraction we use in the next experiments. Sizedetermination has been assessed on the basis of the retention time withrespect to the known sizes and timing of the ladder peaks. The fractionof interest is between 10 and 120 nucleotides (nt) for all the plantsRNA extracts. Synthetic plant small RNA duplexes (FIG. 2) weresynthesized by Sigma Genosys and purified by HPLC. The annealing ofsmall RNA strands and the methylation at 3′ was performed as describedby Sigma oligo synthesis service(http://www.sigmaaldrich.com/life-science/custom-oligos/sirna-oligos.html).Upon arrival the small RNA duplexes were resuspended in RNAse-free waterto the appropriate concentration.

Example 2: Evaluation of sRNAs Effects on the T Cell Proliferation (FIG.3)

In a Mixed Lymphocyte Reaction (MLR) assay, human CD4+ T cells wereco-cultured with allogenic human monocyte-derived DCs in presence or not(positive control) of the different plant sRNA extracts. The MLR is afunctional assay which measures the proliferative response oflymphocytes from one individual (the responder) to lymphocytes fromanother individual (the stimulator). In detail, peripheral bloodmononucleated cells were collected by Ficoll gradient centrifugationfrom healthy donor buffy coat. CD14⁺ monocytes were then isolated by apositive magnetic selection and differentiate in immature DCs in mediumcontaining IL-4 and GM-CSF. After 5 days, such monocyte-derived DCs werecollected and used for the following challenges experiments. For MLR,DCs were co-cultured in the presence of CD4+ T cells with or without thepresence of sRNA extracts. In each experiment (this or the following)sRNAs or miRNAs are complexed with DOTAP, a liposomal transfectionagent, which facilitates nucleic acid entry into the cells (Carvalho etal. TLR3 essentially promotes protective class I-restricted memory CD8⁺T-cell responses to Aspergillus fumigatus in hematopoietic transplantedpatients. Blood. 2012 Jan. 26; 119(4):967-77; Bourquin C et alImmunostimulatory RNA oligonucleotides induce an effective antitumoralNK cell response through the TLR7. J Immunol. 2009 Nov. 15;183(10):6078-86). DOTAP is also added to the control cells to excludecross reactivity). Any sRNA extracts tested, used at the morephysiological concentration retrieved in host biofluids (10 ng/ml,) wereable to attenuate the T cell proliferation (FIG. 3).

Example 3: Evaluation of miRNAs Effects on the Immune Cells Propertiesand their Ability to Respond to an Inflammatory Stimulus

In in vitro experiments, human monocyte-derived DCs were exposed to thepurified miRNAs (or Fragaria vesca pure sRNA extracts) and thenpre-treated DCs were challenged with purified LPS, thelipopolysaccharide layer of bacterial wall. This experiment alloweddetermining the sRNA effect on the activation process and maturation ofthe DC through co-stimulatory molecules analysis by flow cytometry anddetection of soluble factors such as cytokines by DC on culture medium.Co-stimulatory molecules expressions on DC cell surface are markers ofproper activation and maturation of the immune cell. Increasing levelsof these molecules promote DC-T cells contact and antigen presentationthus activating T cells and their survival, priming the proper adaptiveimmune response. Cytokines could enhance the cellular response (i.epro-inflammatory cytokines as IL-6, TNFα, IL-1β, IL-12p70, IL-23, INFγ,IL-17), promote type-2 response and thus favoring antibody production(IL-4, IL-13, IL-5) or down-regulate inflammatory process (IL-10, TGFβ).

In particular, comparing the levels of CD80, CD86, CD83 and the class IIimmuno-histocompatibility complex (MHCII o HLA-DR) of DC pre-exposed tomiRNAs or treated with LPS alone evidence a possible attenuation effectsof the specific miRNAs. In parallel by evaluating the released cytokineprofiles the ability of DCs to properly respond to the stimuli wasassessed. Measuring the level of pro-inflammatory (IL-6, TNFα, IL-1β,IL-12p70) and anti-inflammatory (IL-10) cytokines, allowed appreciationof the immuno-modulatory ability of the specific miRNAs. For all treatedDC, 24 h-IL-1β, TNFα, IL-10 production and 48 h-CD80, CD86, CD83 andMHCII expression were analyzed. The cells were then used in mixedlymphocyte reaction, where treated DCs were co-cultured in the presenceof CD4+ T cells. INFγ production was evaluated on culture supernatantsafter 5 days.

The general experimental design is shown in FIG. 4.

In a first set of experiments dendritic cells (DC) were treated with thefollowing agents, respectively:

-   -   Fragaria vesca specific small duplex miR156 (FvmiR156) (SEQ ID        Nos. 1 and 2; FIG. 2, panel A)    -   Fragaria vesca small duplex miR168 (FvmiR168) (SEQ ID Nos. 3 and        4; FIG. 2, panel B)    -   rice small duplex osamiR168 (SEQ ID Nos. 5 and 6; FIG. 2, panel        C)    -   Fragaria vesca total sRNAs fraction (FvsRNA)    -   small duplex chemically modified green fluorescent protein        sequence (sGFP; SEQ ID Nos. 7 and 8; FIG. 2, panel D) (these        already served as negative control for many groups (see Robbins        M, 2008, Hum Gen Ther));    -   F. vesca total high molecular weight RNA fraction (FvRNA)    -   E. coli LPS (positive control)    -   E. coli LPS after 2 h pre-exposure to the all nucleic acids        above

Several miRNAs concentrations were tested, ranging from thephysiological (10 ng/ml) to the common experimental concentrations fornucleic acids (10 μg/ml, Liu X1 et al. (2010)). MicroRNA-148/152 impairinnate response and antigen presentation of TLR-triggered dendriticcells by targeting CaMKIIα. J Immunol. 15; 185(12):7244-51; Carvalho etal. TLR3 essentially promotes protective class I-restricted memory CD8⁺T-cell responses to Aspergillus fumigatus in hematopoietic transplantedpatients. Blood. 2012 Jan. 26; 119(4):967-77; Bourquin C et alImmunostimulatory RNA oligonucleotides induce an effective antitumoralNK cell response through the TLR7. J Immunol. 2009 Nov. 15;183(10):6078-86). Even if the strongest inhibitory effects were seen atthe highest concentrations, in order to avoid possible saturationeffects of the system the Example showed results using the morephysiological concentration of miRNAs retrieved in host biofluids (FIGS.5 and 6). While the high molecular weight RNA fraction does not affectDCs activation by LPS, plant miRNAs and plant sRNA fraction modulateboth cytokine and co-stimulatory expression.

In particular, plant sRNAs significantly attenuate IL-1β, TNFα and IL-10production by LPS, as shown in FIG. 5. Moreover, FvmiR68 showed thestrongest effects.

In addition, it could be shown that plant sRNAs limit the increase ofCD80, CD86 and CD83 expression (FIG. 6).

In a further set of experiments (FIGS. 7 and 8), in order to investigatethe possible effect of the presence of the methyl group in the plantmiRNA sequence, which is not present in endogenous DC human miRNAs, twohuman miRNAs (miR148, miR155 and its star form miR155*), known to affectDC response to LPS (for a review, Zhan and Wu, Functional regulation ofmonocyte-derived dendritic cells by microRNAs. Protein Cell. 2012 July;3(7):497-507) were used. DCs were treated with the following agents,respectively:

-   -   human miR155    -   human miR155 methylated    -   human miR148    -   human miR148 methylated

The methylated miRNAs were synthetized by adding a methyl group at the2′OH group of the 3′ terminus.

The methyl group did not change the ability of human miRNAs to modulatethe DC response to LPS.

In further experiments to evaluate if not only sRNA fractions but alsosynthetic plant miRNA could modulate T cell proliferation, prior LPStreatment DCs were exposed for 2 h to:

-   -   Fragaria vesca small duplex miR168 (FvmiR168) (SEQ ID Nos. 3 and        4; FIG. 2, panel B)    -   rice small duplex osamiR168 (SEQ ID Nos. 5 and 6; FIG. 2, panel        C)    -   Fragaria vesca total sRNAs fraction (FvsRNA)    -   hsamiR155    -   hsamiR148        and then co-cultured with CD4⁺ T cells. sRNA- and        miRNA-treated-DCs seem to have a lower immunostimulatory ability        since LPS-induced IFN-γ is less produced by T cells (FIG. 9).        MLR assay showed that FvmiR168 and FvsRNA modulate T cell        proliferation (FIG. 10).

The invention claimed is:
 1. A method for treating and/or preventing aninflammatory disease, comprising administering to a patient in needthereof a pharmaceutically effective amount of miR168, wherein theinflammatory disease is characterized by an increase in expression orproduction of IL-1β, TNFα, or IL-10 or is characterized by an increasein CD4+ T cell proliferation, and wherein the miR168 attenuates the CD4+T cell proliferation or attenuates expression or production of IL-1β,TNFα, or IL-10.
 2. A method for preventing an inflammatory disease,comprising administering to a human miR168, wherein said human is ahealthy human, or a human not suffering from said inflammatory disease,wherein the inflammatory disease is characterized by an increase inexpression or production of IL-1β, TNFα, or IL-10 or is characterized byan increase in CD4+ T cell proliferation, and wherein the miR168attenuates the CD4+ T cell proliferation or attenuates expression orproduction of IL-1β, TNFα, or IL-10.
 3. The method of claim 1, whereinsaid miR168 is comprised in a pharmaceutical composition comprisingmiR168 and at least one carrier.
 4. The method of claim 3, wherein saidat least one carrier is selected from saline, sugars, polypeptides,polymers, lipids, creams, gels, water, a glucose solution, apolycationic binding agent, a cationic polypeptide, a hydrophilicpolymer grafted polymer, a non-natural cationic polymer, a cationicpolyacetal, a hydrophilic polymer grafted polyacetal, a ligandfunctionalized cationic polymer, a ligand functionalized-hydrophilicpolymer grafted polymer, a ligand functionalized liposome, abiodegradable histidine-lysine polymer, a biodegradable polyester,poly(lactic acid) (PLA), poly(glycolic acid) (PGA),poly(lactic-co-glycolic acid) (PLGA), a polyamidoamine (PAMAM)dendrimer, a PEGylated PEL, a micelle material, a metal nanoparticle, ananoparticle, a cationic lipid, a cationic micelle, a liposome and anexosome.
 5. The method of claim 3, wherein said pharmaceuticalcomposition further comprises at least one additive, adjuvant, excipientor diluent selected from water, gelatin, vegetable gum, ligninsulfonate,talc, sugar, starch, gum arabic, a vegetable oil, a polyalkylene glycol,a flavoring agent, a preservative, a stabilizer, a emulsifying agent, abuffer, a lubricant, a colorant, a wetting agent, and a filler.
 6. Themethod of claim 1, wherein said miR168 (a) is methylated at the 2′OHgroup at the 3′ terminus, (b) is synthetic miRNA, (c) is purified fromplant, (d) is comprised in a plant sRNA extract, or (e) is F. vescamiR168 or osamiR168.
 7. The method of claim 2, wherein said miR168 (a)is methylated at the 2′OH group at the 3′ terminus, (b) is syntheticmiRNA, (c) is purified from plant, (d) is comprised in a plant sRNAextract, or (e) is F. vesca miR168 or osamiR168.
 8. The method of claim2, wherein said miR168 is comprised in a dietary supplement, food orfood additive.
 9. The method of claim 1, wherein said inflammatorydisease is selected from the group consisting of chronic prostatitis,Glomerulonephritis, Hypersensitivities, Pelvic inflammatory disease,Reperfusion injury, Sarcoidosis, Vasculitis, Interstitial cystitis,normocomplementemic urticarial vasculitis, pericarditis, myositis,anti-synthetase syndrome, scleritis, macrophage activation syndrome,Bechet's Syndrome, PAPA Syndrome, Blau's Syndrome, gout, adult andjuvenile Still's inflammatory disease, cryropyrinopathy, Muckle-Wellssyndrome, familial cold-induced auto-inflammatory syndrome, neonatalonset multisystemic inflammatory disease, familial Mediterranean fever,chronic infantile neurologic, cutaneous and articular syndrome, systemicjuvenile idiopathic arthritis, Hyper IgD syndrome, Schnitzler'ssyndrome, TNF receptor-associated periodic syndrome (TRAPSP),gingivitis, periodontitis, hepatitis, cirrhosis, pancreatitis,myocarditis, vasculitis, gastritis, gout, gouty arthritis, andinflammatory skin disorders, selected from the group consisting ofpsoriasis, atopic dermatitis, eczema, rosacea, urticaria, and acne. 10.The method of claim 2, wherein said inflammatory disease is anautoimmune disease or an allergic disease.
 11. The method of claim 10,wherein (a) said inflammatory disease is an autoimmune disease selectedfrom the group consisting of multiple sclerosis, rheumatoid arthritis,psoriatic arthritis, discoid lupus erythematosus, systemic lupuserythematosus (SLE), ulcerative colitis, Crohn's disease, benignlymphocytic angiitis, autoimmune lymphoproliferative syndrome,sarcoidosis, autoimmune thrombocytopenic purpura, idiopathicthrombocytopenic purpura, pure red cell aplasia, Sjogren's syndrome,rheumatic disease, polymyalgia rheumatica, mixed connective tissuedisease, inflammatory rheumatism, degenerative rheumatism,extra-articular rheumatism, juvenile arthritis, juvenile rheumatoidarthritis, systemic juvenile idiopathic arthritis, arthritis uratica,muscular rheumatism, chronic polyarthritis, reactive arthritis, Reiter'ssyndrome, rheumatic fever, relapsing polychondritis, Raynaud'sphenomenon, vasculitis, cryoglobulinemic vasculitis, ANCA-associatedvasculitis, temporal arteritis, giant cell arteritis, Takayasuarteritis, Behcet's disease, antiphospholipid syndrome, myastheniagravis, autoimmune haemolytic anaemia, Guillain-Barre syndrome, chronicimmune polyneuropathy, chronic inflammatory demyelinatingpolyneuropathy, autoimmune thyroiditis, insulin dependent diabetesmellitus, type I diabetes, Addison's disease, membranousglomerulonephropathy, polyglandular autoimmune syndromes, Goodpasture'sdisease, autoimmune gastritis, autoimmune atrophic gastritis, pemphigus,pemphigus vulgaris, cirrhosis, primary biliary cirrhosis, idiopathicpulmonary fibrosis, myositis, dermatomyositis, juvenile dermatomyositis,polymyositis, fibromyositis, myogelosis, celiac disease, celiac spruedermatitis, immunoglobulin A nephropathy, Henoch-Schonlein purpura,Evans syndrome, atopic dermatitis, psoriasis, psoriasis vulgaris,psoriasis arthropathica, Graves' disease, Graves' ophthalmopathy,scleroderma, systemic scleroderma, progressive systemic scleroderma,diffuse scleroderma, localized scleroderma, Crest syndrome, asthma,allergic asthma, allergy, primary biliary cirrhosis, Hashimoto'sthyroiditis, fibromyalgia, chronic fatigue and immune dysfunctionsyndrome (CFIDS), primary myxedema, sympathetic ophthalmia, autoimmuneinner ear disease, autoimmune uveitis, autoimmune chronic activehepatitis, collagen diseases, ankylosing spondylitis, periarthritishumeroscapularis, panarteritis nodosa, polyarteritis nodosa,chondrocalcinosis, Wegener's granulomatosis, microscopic polyangiitis,bullous skin disorders, pemphigoid, bullous pemphigoid, cicatricialpemphigoid, vitiligo, atopic eczema, eczema, chronic urticaria,autoimmune urticaria, normocomplementemic urticarial vasculitis,hypocomplementemic urticarial vasculitis, alopecia areata, alopeciauniversalis, alopecia totalis, Devic's disease, pernicious anemia,childhood autoimmune hemolytic anemia, idiopathic autoimmune hemolyticanemia, refractory or chronic Autoimmune Cytopenias, Cold agglutinindisease, Neuromyelitis Optica, Stiff Person Syndrome, gingivitis,periodontitis, pancreatitis, myocarditis, gastritis, gout, goutyarthritis, idiopathic pericarditis, anti-synthetase syndrome, scleritis,macrophage activation syndrome, PAPA Syndrome, Blau's Syndrome, adultand juvenile Still's disease, cryopyrin associated periodic syndrome,Muckle-Wells syndrome, familial cold auto-inflammatory syndrome,neonatal onset multisystem inflammatory disease, chronic infantileneurologic cutaneous and articular syndrome, familial Mediterraneanfever, Hyper IgD syndrome, Schnitzler's syndrome, autoimmuneretinopathy, age-related macular degeneration, and TNFreceptor-associated periodic syndrome (TRAPS), or (b) said inflammatorydisease is an allergic disease selected from the group consisting ofallergic respiratory disease, allergic nasal disease, allergic skindisease, allergic ocular disease, such as hay fever, seasonal allergicconjunctivitis, or chronic allergic conjunctivitis, hypersensitivitypneumonitis, contact dermatitis, and food allergy.
 12. The method ofclaim 9, wherein said inflammatory disease is psoriasis.
 13. The methodof claim 2, wherein said inflammatory disease further comprises anincrease in expression or production of at least one of inflammatorybiomarker or wherein the inflammatory disease is characterized by adysregulation of the cytokines or co-receptors in at least one cell ortissue of a patient.
 14. The method of claim 8, wherein said dietarysupplement is formulated for oral delivery and/or wherein the dietarysupplement comprises at least one excipients or at least one carrier fororal consumption.
 15. The method of claim 14, wherein the carrier is aliquid, gel, gelcap, capsule, powder, solid tablet or tea.
 16. Themethod of claim 8, wherein the dietary supplement is provided as apowder or liquid.
 17. The method of claim 8, wherein said food isselected from a beverage, a soup, a dairy product, a nutritional bar, aspread, a prepared food, a packaged food, or an animal feed.
 18. Amethod for treating and/or preventing an inflammatory disease,comprising administering to a patient in need thereof a pharmaceuticallyeffective amount of an RNA which has a sequence selected from thesequences set forth in SEQ ID NOS: 3 and 5, wherein the inflammatorydisease is characterized by an increase in expression or production ofIL-1β, TNFα, or IL-10 or is characterized by an increase in CD4+ T cellproliferation, and wherein the RNA attenuates the CD4+ T cellproliferation or attenuates expression or production of IL-1β, TNFα, orIL-10.
 19. A method for preventing an inflammatory disease, comprisingadministering to a human an RNA which has a sequence selected from thesequences set forth in SEQ ID NOS: 3 and 5, wherein said human is ahealthy human, or a human not suffering from said inflammatory disease,wherein the inflammatory disease is characterized by an increase inexpression or production of IL-1β, TNFα, or IL-10 or is characterized byan increase in CD4+ T cell proliferation, and wherein the RNA attenuatesthe CD4+ T cell proliferation or attenuates expression or production ofIL-1β, TNFα, or IL-10.
 20. The method of claim 4, wherein the cationiclipid is selected from DOTAP, DOPE, DC-Chol/DOPE, DOTMA, and DOTMA/DOPE.21. The method of claim 2, further comprising administering a cationiclipid carrier, wherein the cationic lipid is selected from DOTAP, DOPE,DC-Chol/DOPE, DOTMA, and DOTMA/DOPE.
 22. The method of claim 18, furthercomprising administering a cationic lipid carrier, wherein the cationiclipid is selected from DOTAP, DOPE, DC-Chol/DOPE, DOTMA, and DOTMA/DOPE.23. The method of claim 19, further comprising administering a cationiclipid carrier, wherein the cationic lipid is selected from DOTAP, DOPE,DC-Chol/DOPE, DOTMA, and DOTMA/DOPE.